The present invention provides a method for treating neurodegenerative disorders. More particularly, a method for treating neurodegenerative disorders (e.g., Alzheimer""s disease) by inhibiting the interaction of amyloid beta with alpha-7 nicotinic acetylcholine receptors.
Neurodegenerative disorders such as Alzheimer""s disease (AD) and Parkinson""s disease (PD) afflict humanity with great suffering and financial loss. AD is characterized by neurofibrillary tangles, neuritic plaques, and neuronal cell death. AD appears as either the familial, early onset ( less than 60 yrs) or late-onset ( greater than 60 yrs) forms, with the latter being more prevalent. AD is the major cause of age-related dementia and cognitive impairment (Wisniewski, T.; Ghiso, J.; Frangione, B. Neurobiol. of Disease 1997, 4, 313-328). The amyloid precursor protein (APP), xcex2-amyloid1-40 (Axcex21-40), and xcex2-amyloid1-42 (Axcex21-42) are keenly involved in the pathology of AD. The Axcex2 peptides are derived from APP by proteolytic processing. Dramatic evidence implicating the Axcex2 peptides, particularly Axcex21-42, in AD comes from various recently identified mutations accounting for certain types of inherited AD. Such mutations in the presenilin (PS1 and PS2) genes are probably the cause of the most frequent form of familial, early-onset AD (Rogaev, E. I. Molecular Biology 1998, 32, 58). In these cases, as with APP mutations, more Axcex21-42 is observed relative to Axcex21-40. Extensive studies have shown that Axcex21-42 has a greater ability than Axcex21-40 to aggregate into the amyloid fibrils that constitute the plaques characteristic of AD (Lansbury, P. T., Jr. Accts. Chem. Res. 1996, 29, 317). Even though Axcex21-40 is generally present to a much larger degree in the cerebrospinal fluid than Axcex21-42, it is Axcex21-42 which is the major Axcex2 peptide found in AD plaques.
The Axcex2 peptides can inhibit cholinergic neurotransmitter function independent of neurotoxicity (Auld, D. S.; Kar, S.; Quirion, R. Trends Neurosci. 1998, 21, 43). Axcex2 peptides bind to a number of natural substances such as apoE3, apoE4, apoJ, transthyretin, and albumin. In addition, Axcex2 has been reported to interact with a membrane-bound receptor for advanced glycation end products and to the class A scavenger receptor (SR) associated with the production of reactive oxygen species. Stimulation of the alpha-7 subtype of the nicotinic acetylcholine receptors (nAChRs) can protect neurons against Axcex2 cytotoxicity (Kihara, T. et al. Ann. Neurol. 1997, 42, 159). Also, a set of compounds that activate nAChRs, especially of the alpha-7 subtype, have been found to have in vivo activity in models of cognition enhancement (U.S. Pat. No. 5,741,802, issued Apr. 21, 1998).
We now describe specific binding of Axcex21-40 and Axcex21-42 to the alpha-7 subtype of nAChRs. This new finding has broad ramifications for the etiology and treatment of AD. nAChRs are members of the ligand-gated ion channel family and appear to be formed from five protein subunits associating together around a central pore (Lindstrom, J. Molecular Neurobiology 1997, 15, 193). These subunits include xcex11-xcex19, xcex21-xcex24, xcex3, xcex4, and xcex5. The xcex17 subtype forms functional homomers which bind to xcex1-bungarotoxin, a 75-amino acid peptide, with high affinity (0.65-1.7 nM Kd) and nicotine with relatively low affinity (ca. micromolar Kd) (Holladay, M. W.; Dart, M. J.; Lynch, J. K. J. Med. Chem. 1997, 40, 4169).
Compounds which block the aggregation of Axcex2 peptides are potentially useful drugs for the treatment of AD. For example, rifampicin inhibits Axcex2 aggregation and neurotoxicity and may show an effect in vivo in diminishing plaque burden when compared with age-matched controls (Tomiyama, T. et al. J. Biol. Chem. 1996, 271, 6839). In order to block the interaction of the Axcex2 peptides with xcex17 nAChRs, compounds can be found to either bind to xcex17 nAChRs, to Axcex2 itself, or to both. Any of these mechanisms of action would be expected to provide significant protection against Axcex2-mediated neurotoxicity and inhibition of cholinergic functioning mediated by nAChRs and be extremely useful for the treatment of AD. The binding of Axcex21-42 to alpha-7 nAChRs provides a seed for crystallization or deposition of Axcex2 into insoluble deposits, which have the potential to grow into the fibrillar amyloid deposits characteristic of AD. Therefore, blocking the interaction of Axcex21-42 with alpha-7 nAChRs should reduce the amount of insoluble aggregated Axcex2 that is formed, and thus prevent the neurotoxicity and pathology associated with such aggregated amyloid deposits.
Accordingly, it is an object of the invention to provide a method for treating neurodegenerative disorders by inhibiting the binding of amyloid beta peptides to alpha-7 nicotinic acetylcholine receptors. It is a further object of the invention to provide a method for treating Alzheimer""s disease and/or for slowing the progression of Alzheimer""s disease by inhibiting the binding of amyloid beta peptides to alpha-7 nicotinic acetylcholine receptors. Another object of the invention is to provide a predictive method, a method for diagnosis, a method to monitor prognosis, a method to monitor the progression, and a method to monitor the therapeutic efficacy for any therapeutic intervention used in Alzheimer""s disease. Still another object of the invention is to provide a method for identifying compounds which inhibit the binding of Axcex2 peptides with xcex17 nAChRs, either by binding to Axcex2 peptides or to xcex17 nAChRs.
The present invention is directed to a method of treating a neurodegenerative disorder in a subject (preferably, a human) in need thereof which comprises administering to the subject an amount of a compound effective to inhibit the binding of an amyloid beta peptide, preferably Axcex21-42, to alpha-7 nAChRs, preferably, human alpha-7 nAChRs. Since alpha-8 and alpha-9 nAChRs are similar with respect to structure and function to alpha-7 nAChRs, it is possible that blocking the interaction of xcex2-amyloid with alpha-8 and alpha-9 nAChRs would have therapeutic benefit as well.
Neurodegenerative disorders included within the methods of the present invention include, but are not limited to, Alzheimer""s disease, Pick""s disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson""s-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington""s disease, Parkinson""s disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette""s disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy""s disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sach""s disease, Sandhoff disease, familial spastic disease, Wohifart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob, Gerstmann-Strxc3xa4ussler-Scheinker disease, Kuru and fatal familial insomnia).
Other conditions also included within the methods of the present invention include age-related dementia and other dementias and conditions with memory loss including vascular dementia, diffuse white matter disease (Binswanger""s disease), dementia of endocrine or metabolic origin, dementia of head trauma and diffuse brain damage, dementia pugilistica and frontal lobe dementia. Also other neurodegenerative disorders resulting from cerebral ischemia or infaction including embolic occlusion and thrombotic occlusion as well as intracranial hemorrhage of any type (including, but not limited to, epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (including, but not limited to, contusion, penetration, shear, compression and laceration).
Preferably, the neurodegenerative disorder is selected from Alzheimer""s disease, Parkinson""s disease, Tourette""s syndrome, amyotrophic lateral sclerosis, age-related memory loss, senility and age-related dementia, most preferably, the neurodegenerative disorder is Alzheimer""s disease. Because, most preferably, the neurodegenerative disorder is Alzheimer""s disease, also defined as an amyloidosis, other conditions within the methods of the present invention include other amyloidosis which share features including, but not limited to, hereditary cerebral angiopathy, nonneuropathic hereditary amyloid, Down""s syndrome, macroglobulinemia, secondary familial Mediterranean fever, Muckle-Wells syndrome, multiple myeloma, pancreatic- and cardiac-related amyloidosis, chronic hemodialysis arthropathy, and Finnish and Iowa amyloidosis.
In one embodiment of the invention is a method of treating and/or preventing dementia in an Alzheimer""s patient (as well as a method for treating and/or preventing other clinical manifestations of Alzheimer""s disease that include, but are not limited to, cognitive and language deficits, apraxias, depression, delusions and other neuropsychiatric symptoms and signs, and movement and gait abnormalities) which comprises administering to the subject a therapeutically effective amount of a compound to inhibit the binding of an amyloid beta peptide (preferably, Axcex21-42) with nAChRs, preferable alpha-7 nAChRs, most preferably, human alpha-7 nAChRs.
In a second embodiment of the invention is a method of improving memory and/or mental status and/or of halting the progression of mental deterioration in an Alzheimer""s disease patient which comprises administering to the subject a therapeutically effective amount of a compound to inhibit the binding of an amyloid beta peptide (preferably, Axcex2-1-42) with nAChRs, preferably alpha-7 nAChRs, most preferably, human alpha-7 nAChRs.
Preferably, the compound used in the methods of treating neurodegenerative disorders, treating and/or preventing Alzheimer""s disease, and improving memory and/or halting the progression of mental deterioration in an Alzheimer""s disease patient is not estrogen, raloxifene, droloxifene, tamoxifen, idoxifene or levomeloxifene; more preferably, the compound is not estrogen or a selective estrogen receptor modulator (SERM). A SERM is an estrogen receptor ligand that exhibits estrogen agonist activity in the cardiovascular system, CNS and bone, and estrogen antagonist activity in reproductive tissues, such as breast and uterus.
Also included in the invention is the use of a compound which inhibits the binding of an amyloid beta peptide (preferably Axcex21-42) to an alpha-7 nAChR (preferably, a human alpha-7 nAChR) in the preparation of a medicament for the treatment of a neurodegenerative disorder in a subject (preferably, a human) in need thereof.
Another illustration of the invention is the use of a compound which inhibits the binding of an amyloid beta peptide (preferably Axcex21-42) to alpha-7 nAChRs (preferably, human alpha-7 nAChRs) in the preparation of a medicament for: a) improving memory, b) halting the progression of the mental deterioration seen in Alzheimer""s disease patients, c) treating dementia, d) preventing dementia in an Alzheimer""s patient, and e) treating and/or preventing other clinical manifestations of Alzheimer""s disease that include, but are not limited to, cognitive and language deficits, apraxias, depression, delusions and other neuropsychiatric symptoms and signs, and movement and gait abnormalities in an Alzheimer""s patient.
In another aspect of the invention is a compound of the formula I: 
wherein
R1 is hydrogen or C1-C4 alkyl;
R2 is selected from hydrogen, C1-C6 alkyl, aryl or C7-C10 aralkyl;
R3 is selected from hydrogen, C1-C6 alkyl, C3-C10 alkenyl, C3-C8 cycloalkylC1-C6 alkyl, C1-C6 alkoxycarbonylC1-C6 alkyl, C1-C6 alkylthio, heteroarylC1-C4 alkyl, unsubstituted or substituted aryl or unsubstituted or substituted C7-C10 aralkyl wherein the substituent on the aryl or aralkyl are one or more substituents independently selected from the group consisting of halogen, hydroxy, C1-C6 alkyl and unsubstituted or substituted C1-C6 alkoxy wherein the substituents on the alkoxy are one or more substituents independently selected from amino, C1-C6 alkylamino, C1-C6 dialkylamino, pyrrolidinyl, piperidinyl, azepinyl or morpholinyl; or R2 and R3, together with the nitrogen to which they are attached, form a five or six-membered heterocyclic ring selected from pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl or piperazinyl;
R4 is C1-C6 alkyl, aryl, or C7-C10 aralkyl; and
R5 and R6 are each independently selected from hydrogen, C1-C6 alkyl, C3-C10 alkenyl, C1-C8 alkylcarbonyl, or diphenylphosphinyl; and pharmaceutically acceptable salts and prodrugs thereof.
In preferred compounds of formula I,
R1 is hydrogen;
R2 is selected from hydrogen or C1-C4 alkyl;
R3 is selected from C1-C4 alkyl, C3-C10 alkenyl, C5-C6 cycloalkylC1-C46 alkyl, C1-C6 alkoxycarbonylC1-C4 alkyl, C1-C6 alkylthio, heteroarylC1-C4 alkyl, or unsubstituted or substituted C7-C10 aralkyl wherein the substituent on the aralkyl are one or two substituents independently selected from the group consisting of halogen, hydroxy, C1-C4 alkyl and unsubstituted or substituted C1-C4 alkoxy wherein the substituents on the alkoxy are one or two substituents independently selected from amino, C1-C4 alkylamino, C1-C4 dialkylamino, pyrrolidinyl, or piperidinyl; or
R2 and R3, together with the nitrogen to which they are attached, form a morpholinyl ring;
R4 is C1-C4 alkyl; and
R5 and R6 are each independently selected from hydrogen, C1-C4 alkyl, C3-C6 alkenyl, C1-C6 alkylcarbonyl, or diphenylphosphinyl.
In a subclass of compounds of formula I are compounds having the formula 
wherein
R1 is hydrogen or C1-C4 alkyl;
R2 and R3 are each independently selected from hydrogen, C1-C6 alkyl, aryl or C7-C10 aralkyl; and
R4 is C1-C6 alkyl, aryl, or C7-C10 aralkyl;
and pharmaceutically acceptable salts thereof.
Compounds of formula I are novel compounds which block the interaction of beta-amyloid with alpha-7 nAChRs. More specifically, the compounds of formula I inhibit the binding of Axcex21-42with human alpha-7 nAChRs by binding to Axcex21-42. The orientation between the nitrogen atom and R4 on the appropriate ring can be either cis or trans. Preferably, the compound is 5,8-dihydroxy-trans-2-di(N-propylamino)-3-methyl-1,2,3,4-tetrahydronaphthalene, and pharmaceutically acceptable salts thereof.
Other compounds useful in the methods of the present invention inhibit the binding of Axcex21-42 with human alpha-7 nAChRs by binding to human alpha-7 nAChRs directly. An example of such a compound which binds to human alpha-7 nAChRs is xcex1-bungarotoxin.
The present invention provides methods of treating neurodegenerative disorders by inhibiting the binding of amyloid beta peptides to alpha-7 nAChRs. Neurodegenerative disorders included within the methods of the present invention include, but are not limited to, Alzheimer""s disease, Pick""s disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson""s-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington""s disease, Parkinson""s disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette""s disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (Kennedy""s disease), primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sach""s disease, Sandhoff disease, familial spastic disease, Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressive multifocal leukoencephalopathy, and prion diseases (including Creutzfeldt-Jakob, Gerstmann-Strxc3xa4ussler-Scheinker disease, Kuru and fatal familial insomnia).
Other conditions also included within the methods of the present invention include age-related dementia and other dementias and conditions with memory loss including vascular dementia, diffuse white matter disease (Binswanger""s disease), dementia of endocrine or metabolic origin, dementia of head trauma and diffuse brain damage, dementia pugilistica and frontal lobe dementia. Also other neurodegenerative disorders resulting from cerebral ischemia or infaction including embolic occlusion and thrombotic occlusion as well as intracranial hemorrhage of any type (including, but not limited to, epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (including, but not limited to, contusion, penetration, shear, compression and laceration).
Preferably, the neurodegenerative disorder is selected from Alzheimer""s disease, Parkinson""s disease, Tourette""s syndrome, amyotrophic lateral sclerosis, age-related memory loss, senility and age-related dementia, most preferably, the neurodegenerative disorder is Alzheimer""s disease. Because, most preferably, the neurodegenerative disorder is Alzheimer""s disease, also defined as an amyloidosis, other conditions within the methods of the present invention include other amyloidosis which share features including, but not limited to, hereditary cerebral angiopathy, nonneuropathic hereditary amyloid, Down""s syndrome, macroglobulinemia, secondary familial Mediterranean fever, Muckle-Wells syndrome, multiple myeloma, pancreatic- and cardiac-related amyloidosis, chronic hemodialysis arthropathy, and Finnish and Iowa amyloidosis.
The terms xe2x80x9camyloid betaxe2x80x9d, xe2x80x9camyloid beta peptidexe2x80x9d or xe2x80x9cbeta-amyloidxe2x80x9d as used herein, refer to amyloid beta peptides and include the Axcex21-40, Axcex21-42 and Axcex21-43 peptides and their fragments. Examples of fragments of amyloid beta peptides that have been shown to have biological activity and are useful in the methods of the present invention include, but not limited to, fragment 1-28, and fragment 25-35 (e.g., Yatin S M, Aksenov M, Butterfield D A. Neurochem Res 1999 Mar;24(3):427-35; Hirakura Y, Satoh Y, Hirashima N, Suzuki T, Kagan B L, Kirino Y. Biochem Mol Biol Int 1998 Nov; 46(4):787-94; Mazziotti M, Perlmutter D H. Biochem J 1998 Jun 1;332 (Pt 2):517-24; Perovic S, Bohm M, Meesters E, Meinhardt A, Pergande G, Muller W E. Mech Ageing Dev 1998 Mar 16;101 (1-2):1-19; Muller W E, Eckert G P, Scheuer K, Cairns N J, Maras A, Gattaz W F. Amyloid 1998 Mar;5(1):10-5; Butterfield D A, Martin L, Carney J M, Hensley K. Life Sci 1996;58(3):217-28; Forloni G, Lucca E, Angeretti N, Della Torre P, Salmona M. J Neurochem 1997 Nov;69(5):2048-54; Heese K, Hock C, Otten U. J Neurochem 1998 Feb;70(2):699-707; Blanchard B J, Konopka G, Russell M, Ingram V M. Brain Res 1997 Nov 21 ;776(1-2):40-50; Wu A, Derrico C A, Hatem L, Colvin R A. Neuroscience 1997 Oct;80(3):675-84; Muller W E, Romero F J, Perovic S, Pergande G, Pialoglou P. J Neurochem 1997 Jun;68(6):2371-7; Suh Y H. J Neurochem 1997 May;68(5):1781-91; Parpura-Gill A, Beitz D, Uemura E. Brain Res 1997 Apr 18;754(1-2):65-71; Fletcher T G, Keire D A. Protein Sci 1997 Mar;6(3):666-75; Scorziello A, Meucci O, Calvani M, Schettini G. Neurochem Res 1997 Mar;22(3):257-65); Miguel-Hidalgo J J, Vecino B, Fernandez-Novoa L, Alvarez A, Cacabelos R. Eur Neuropsychopharmacol 1998 Aug;8(3):203-8; Maneiro E, Lombardi V R, Lagares R, Cacabelos R. Methods Find Exp Clin Pharmacol 1997 Jan-Feb; 19(1):5-12).
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
The term xe2x80x9calkylxe2x80x9d shall mean straight or branched chain alkanes of one to ten carbon atoms, or any number within this range. For example, alkyl radicals include, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 3-(2-methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-hexyl and 2-methylpentyl. Alkoxy radicals are oxygen ethers formed from the previously described straight or branched chain alkyl groups. Cycloalkyl groups contain 3 to 8 ring carbons and preferably 5 to 7 carbons. Similarly, alkenyl and alkynyl groups include straight and branched chain alkenes and alkynes having 2 to 10 carbon atoms, or any number within this range.
The term xe2x80x9carylxe2x80x9d indicates aromatic groups such as phenyl and naphthyl.
The term xe2x80x9cC7-C10 aralkylxe2x80x9d means an alkyl group substituted with an aryl group wherein the total number of carbon atoms is between 7 and 10 (e.g., benzyl, phenylethyl, phenylpropyl).
The term xe2x80x9cheteroarylxe2x80x9d as used herein represents an unsubstituted or substituted stable five or six membered monocyclic aromatic ring system or an unsubstituted or substituted nine or ten membered benzo-fused heteroaromatic ring system or bicyclic heteroaromatic ring system which consists of carbon atoms and from one to four heteroatoms selected from N, O or S, and wherein the nitrogen or sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroaryl group may be attached at any heteroatom or carbon atom that results in the creation of a stable structure. Examples of heteroaryl groups include, but are not limited to pyridyl, pyridazinyl, thienyl, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl, benzopyrazolyl, indolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl adeninyl or quinolinyl. Prefered heteroaryl groups include pyridyl, pyrrolyl, pyrazinyl, thiadiazolyl, pyrazolyl, thienyl, triazolyl and quinolinyl.
The term xe2x80x9cN(CH2)5xe2x80x9d means a piperidinyl group.
The term xe2x80x9ccC6H11xe2x80x9d refers to a cyclohexyl group.
When a particular group is xe2x80x9csubstitutedxe2x80x9d (e.g., aryl, aralkyl), that group may have one or more substituents, preferably from one to five substituents, more preferably from one to three substituents, most preferably from one to two substituents, independently selected from the list of substituents.
Under standard nomenclature used throughout this disclosure, the terminal portion of the designated side chain is described first, followed by the adjacent functionality toward the point of attachment. Thus, for example, a xe2x80x9cphenylC1-C6 alkylamidoC1-C6alkylxe2x80x9d substituent refers to a group of the formula 
Compounds which are useful in the methods of the present invention for inhibiting the interaction of Axcex21-40 and Axcex21-42 to the alpha-7 subtype of nAChRs for either the purpose of direct therapeutic intervention or in order to screen for compounds which act via this mechanism include compounds of formula I, especially 5,8-dihydroxy-trans-2-di(N-propylamino)-3-methyl-1,2,3,4-tetrahydronaphthalene (Compound 9), (xe2x88x92)-nicotine, (rac)-epibatidine, xcex1-bungarotoxin, and pharmaceutically acceptable salts thereof.
Certain peptide stretches of the human alpha-7 nAChR bind to amyloid beta and can be used in place of the alpha-7 nAChR together with amyloid beta for the purpose of screening libraries to find compounds which block the interaction of amyloid beta and the human alpha-7 nAChR. Included among these peptide stretches of the human alpha-7 nAChR are alpha-7 nAChR193-224 and smaller peptides derived thereof as listed in the Table.
The standard one-letter code for the amino acids has been employed for the compounds. This code is listed in Lehninger, A. I. xe2x80x9cBiochemistryxe2x80x9d Second Edition, Worth Publishers, Inc., New York, 1976, p 73-75.
For use in medicine, the salts of the compounds of this invention refer to non-toxic xe2x80x9cpharmaceutically acceptable salts.xe2x80x9d Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
The present invention includes within its scope prodrugs of the compounds of this invention. A prodrug is inactive as administered, but becomes activated in vivo. The prodrug is converted to the parent drug chemically or by specific enzyme(s). Higuchi, T.; Stella, V., Eds. xe2x80x9cPro-Drugs as Novel Drug Delivery Systemsxe2x80x9d; American Chemical Society: Washington, D.C., 1976. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term xe2x80x9cadministeringxe2x80x9d shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, ed. H. Bundgaard, Elsevier, 1985.
Where the compounds according to this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
(xe2x88x92)-Nicotine is (xe2x88x92)-1-methyl-2-(3-pyridinyl)pyrrolidine and is readily available from Sigma Chemical Company. 
(+/xe2x88x92)-Epibatidine is exo-(+/xe2x88x92)-2-(6-chloro-3-pyridinyl)-7-azabicyclo[2.2.1]heptane and is readily available from Sigma Chemical Company. 
xcex1-Bungarotoxin is a 74 amino acid peptide which is commercially available from Research Biochemicals Inc. xcex1-Bungarotoxin and its amino acid sequence are described in Lee, C. Y. Annu. Rev. Pharmacol. 1972, 12, 265-281.
125I-Axcex21-40, fluo-Axcex21-40, and anti-alpha-7 nAChR antibodies are commercially available Amersham Pharmacia Biotech, Advanced Bioconcepts and Research Biochemicals International, respectively.
125I-xcex1-bungarotoxin is commercially available from Amersham Pharmacia Biotech.
The present invention therefore provides a method of treating a neurodegenerative disorder, which comprises administering any of the compounds as defined herein in a quantity effective to treat the neurodegenerative disorder. Preferably, the compound is not estrogen, raloxifene, droloxifene, tamoxifen, idoxifene or levomeloxifene; more preferably, the compound is not estrogen or a selective estrogen receptor modulator (SERM). The compound may be administered to a patient afflicted with a neurodegenerative disorder by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal, buccal, intracerebral and other parenteral routes. The quantity of the compound which is effective for treating a neurodegenerative disorder is between 0.01 mg per kg and 10 mg per kg of subject body weight.
The method of treating neurodegenerative disorders described in the present invention may also be carried out using a pharmaceutical composition comprising any of the compounds as defined herein and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain between about 0.5 mg and 200 mg of the compound, and may be constituted into any form suitable for the mode of administration selected. Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, caplets, and powders, and liquid forms, such as solutions, syrups, elixers, and suspensions. Forms useful for intracerebral and other parenteral routes of administration include sterile solutions, emulsions and suspensions.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, body weight, diet, physical activity and time of administration, and associated co-morbidities and clinical conditions will result in the need to adjust dosages.
The present invention also provides diagnostic tools useful for diagnosing Alzheimer""s disease. Alzheimer""s disease (AD) exhibits neuropathological abnormalities in the olfactory system located in the nasal cavity. These include the presence of dystrophic neurites that exhibit immunoreactivity for tau, neurofilaments, apolipoprotein E and other proteins, abnormal tau protein, increase in superoxide dismutase, and beta-amyloid deposition in the primary sensory (olfactory receptor) cells and nerve fibres of the nasal mucosa tissue (Arnold et al., Ann N Y Acad Sci 1998 Nov 30;855:762-75; Hock et al., Eur Neurol 1998 Jul;40(1):31-6; Johnson et al., Neurobiol Aging 1994 Nov-Dec; 15(6):675-80; Kulkarni-Narla et al., Exp Neurol 1996 Aug;140(2):115-25; Lee et al., Exp Neurol 1993 May;121(1):93-105; Tabaton et al., Neurology 1991 Mar;41 (3):391-4; Talamo et al., Ann N Y Acad Sci 1991;640:1-7; Yamagishi et al., Ann Otol Rhinol Laryngol 1998 May;107(5 Pt 1):421-6; Yamagishi et al., Nippon Jibiinkoka Gakkai Kaiho 1994 Jan;97(1):51-60). These observations recapitulate the neuropathological profile and neurodegenerative abnormalities (e.g., cytoskeletal changes, protein immunoreactivity and beta-amyloid deposition) observed in central nervous system neurons from AD patients. Routine access to these sensory neurons and fibers can be done with nasal biopsy in AD patients (e.g., Feron et al., Arch Otolaryngol Head Neck Surg 1998 Aug;124(8):861-6).
Olfactory neuroblasts (olfactory neurons obtained by biopsy and placed in primary cell culture) from AD patients produce carboxy terminal amyloid precursor protein (APP) fragments that contain beta-amyloid (A-beta)(Crino et al., Ann Otol Rhinol Laryngol 1995 Aug;104(8):655-61). Crino et al. showed labeling of A-beta in the basal third of the olfactory neuroepithelium and in axons projecting through the lamina propria of AD patients. Thioflavin-S staining that detects amyloid deposition was also observed in the basal third of the olfactory neuroepithelium from AD patients. Alpha 7 nicotinic acetylcholine receptors are present in olfactory neurons probably including olfactory receptor cells in the nasal cavity (Alkondon et al., Neurosci Lett 1994 Aug 1;176(2):152-6; Alkondon et al., Eur J Neurosci 1997 Dec;9(12):2734-42; Bouvet et al., Neurosci Res 1988 Feb;5(3):214-23; Edwards et al., Experientia 1987 Aug 15;43(8):868-73; Edwards et al., Experientia 1988 Mar 15;44(3):208-11; Seguela et al., J Neurosci 1993 Feb;13(2):596-604).
Beta-amyloid peptide increases cytosolic-free Ca2+ in AD lymphoblasts (Ibarreta et al., Alzheimer Dis Assoc Disord 1997 Dec;11(4):220-7), and elevates mitogen-induced Ca2+ responses in freshly prepared human lymphocytes (Eckert et al., Life Sci 1994;55(25-26):2019-29). Amyloid precursor protein (APP) can be induced on the cell surface of human lymphocytes upon stimulation (Bullido et al., Biochim Biophys Acta 1996 Aug 21;1313(1):54-62) and increased APP-770 isoform occurs in lymphocytes from AD patients (Ebstein et al., Brain Res Mol Brain Res 1996 Jan;35(1-2):260-8). Lymphoblastoid cells from patients with early-onset and late-onset familial AD show increased expression of beta-APP mRNA and protein (Matsumoto et al., Eur J Biochem 1993 Oct 1;217(1):21-7). Lymphocytes from AD patients also exhibit an increased mRNA level for alpha 7 nicotinic receptor (Hellstrom-Lindahl et al., Brain Res Mol Brain Res 1999 Mar 20;66(1-2):94-103)
Based on the information described above, we propose that the analysis of the alpha 7 nicotinic acetylcholine receptorxe2x80x94beta amyloid peptides interaction in circulating blood cells and olfactory neuroepithelial neurons/neuronal processes or olfactory neuroblasts obtained from AD patients could be used as AD diagnostic tools, markers of AD progression and prognosis, and markers of therapeutic efficacy for any intervention or treatment targeting AD.
Thus, the present invention provides methods for diagnosing Alzheimer""s disease, monitoring the progression and prognosis of Alzheimer""s disease and/or monitoring the therapeutic efficacy of any intervention or treatment of Alzheimer""s disease comprising:
(a) obtaining a test sample from a subject wherein the test sample comprises circulating blood cells and/or olfactory neuroepithelial neuronal cell bodies or their neuronal processes (i.e., dendrites and axon of a nueron); and
(b) analyzing the test sample for interaction of an amyloid beta peptide (including, but not limited to, Axcex21-40, Axcex21-42 and Axcex21-43 peptides and their fragments) with alpha-7 nicotinic acetylcholine receptors.
The compounds of formula I, such as 5,8-Dihydroxy-trans-2-di(N-propylamino)-3-methyl-1,2,3,4-tetrahydronaphthalene (Compound 9), are made according to the procedures described in the Schemes and Examples which follow.
Abbreviations used in the instant specification, particularly the Schemes and Examples, are as follows:
Ac=acetyl
Ach=acetylcholine
AcOH=acetic acid
BSA=bovine serum albumin
DMF=N,N-dimethyl formamide
DMSO=dimethyl sulfoxide
Et3N=triethyl amine
EtOAc=ethyl acetate
FCS=fetal calf serum
i-Pr=isopropyl
Me=methyl
Mel=methyl iodide
nAChR=nicotinic acetylcholine receptor
Ph=phenyl
PCC=pyridinium chlorochromate
TEA=triethyl amine
THF=tetrahydrofuran
TLC=thin layer chromatography 
Compounds of the invention can be prepared as shown in Scheme 1. First a Diels-Alder reaction is performed on benzophenone (1) with a suitable diene such as isoprene to give a dione such as (2). Then, base-catalyzed isomerization of (2) affords a 1,4-dihydroxyphenyl compound (3), which is converted to dimethyl ether (4). Hydroboration followed by oxidation give alcohol (5), which is then oxidized to give ketone (6). Reductive amination with an amine such as propyl amine is then carried out using a suitable hydride reducing agent such as sodium cyanoborohydride, to give, for example, compound 7. This material can then be subjected to a reductive amination reaction such as with propionaldehyde as shown to yield (8). Compound (8) is then treated with HBr in acetic acid to cleave the methyl ethers and form dihydroxy compound (9). Alternatively, BBr3 may be used to cleave the methyl ethers and form dihydroxy compound (9). Moreover, a deficiency of BBr3 may be used to provide compounds in which only one of the methyl ethers has been removed to afford compounds with one hydroxy and one methoxy group. 
A further means of preparing compounds of the present invention is illustrated in Scheme 2. A suitable propargyl alcohol, such as 3-hydroxy-3-methyl-1-butyne (10, R4=Me) is dehydrated to give an enyne such as compound 11. This material is then subjected to an aminomercuration reaction to afford a 2-amino-1,4-butadiene such as compound (12). Diels-Alder reaction of compound (12) with benzophenone (1) gives (13) which can be isomerized as in Scheme 1 with base to give the 1,5-dihyroxy compound (14). Reduction of the double bond of (14) catalytically with hydrogen and palladium on carbon, such as 10% palladium on carbon, yields compounds of the invention such as (15).
Compounds of type 9 can be treated with a base such as triethylamine in a suitable solvent such as dioxane or methylene chloride along with electrophiles such as acid halides, phosphinyl halides, or alkyl halides to give the products of substitution on one or both of the phenolic hydroxyls. Such compounds can be active by themselves, or serve as prodrugs for compound 9.
The following Examples are set forth to aid in the understanding of the invention, and are not intended and should not be construed to limit in any way the invention set forth in the claims which follow thereafter.